Target-dependent polymerisation of oligonucleotides
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Solution Overview
Problem
Existing Hybridization Chain Reaction (HCR) technologies face challenges such as high background signals, irreversibility, difficulty in controlling reaction kinetics, and limited reproducibility, making them unsuitable for rapid, industrial, and routine applications.
Innovation Solution
Designing HCR oligonucleotide probes with multifunctional hairpin loops that control hybridization specificity and free energy, enabling rapid and accurate detection of target nucleic acids using kits that do not rely on toe-holding, and employing constructs with signal generating components like FRET and chemiluminescence for enhanced sensitivity and control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional HCR is used to detect target nucleic acid, then signal amplification is achieved, but background signals are high and reliability deteriorates
Solution Approach 1:
The hairpin probes are pre-designed with specific secondary structures that remain closed and inactive until triggered by the target nucleic acid. This preliminary configuration prevents background signals by ensuring probes only become active when needed, improving both detection accuracy and reproducibility
Solution Approach 2:
The invention modifies the thermodynamic parameters of the HCR system by designing hairpin probes with specific stem-loop structures and controlling their melting temperatures. This allows precise control over probe activation thresholds, reducing background noise while maintaining signal amplification capability
2Productivity
If traditional HCR with toe-holding is used, then signal amplification occurs, but reaction kinetics control becomes difficult and device complexity increases
Solution Approach 1:
The invention removes the toe-holding element from the traditional HCR mechanism, simplifying the probe design to essential hairpin structures. This extraction eliminates the complexity of controlling toe-holding initiation while preserving the core signal amplification functionality through target-triggered hairpin opening
Solution Approach 2:
The hairpin probes automatically regulate their own activation through target-dependent conformational changes. The probes self-assemble and self-regulate the reaction kinetics without requiring external control mechanisms, simplifying the overall system while maintaining amplification efficiency
3Productivity
If rapid detection is implemented with shortened hybridization time, then productivity improves, but measurement precision may deteriorate
Solution Approach 1:
The hairpin probe structure creates a curved, pre-organized configuration that positions the binding region optimally for target interaction. This structural curvature facilitates rapid hybridization by reducing the entropic penalty of binding, enabling fast detection without sacrificing accuracy
Solution Approach 2:
The probes are pre-configured in closed hairpin states with binding regions sequestered and ready for rapid activation. This preliminary organization allows immediate target binding upon encounter, achieving both speed and precision without requiring extended hybridization times
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The solution provides rapid, accurate, and reproducible nucleic acid detection within two hours, preferably under one hour, with hybridization times as short as 30 minutes, suitable for automation and multiplexing, and applicable to various sample types.
Implementation Method 1
on binding of the target nucleic acid to the Target Initiation probe the stem region of the Target Initiation probe opens and permits hybridisation to the loop region of the first Chain Loop probe
Implementation Method 2
triggers a cascade reaction of subsequent hybridisations, more precisely, a cascade of toehold-mediated strand displacements, leading to the formation of a long nicked double-stranded DNA
Implementation Method 3
signal generating components like FRET and chemiluminescence for enhanced sensitivity
Implementation Method 4
signal generating components like FRET and chemiluminescence for enhanced sensitivity
Data Source
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AI summary
The present invention provides kits and methods for the rapid and sensitive detection of a target nucleic acid through target initiated polymerisation.